Neu4, a Novel Human Lysosomal Lumen Sialidase, Confers NormalPhenotype to Sialidosis and Galactosialidosis Cells*

Received for publication, April 23, 2004, and in revised form, June 10, 2004Published, JBC Papers in Press, June 22, 2004, DOI 10.1074/jbc.M404531200

Volkan Seyrantepe‡§, Karine Landry‡, Stephanie Trudel‡, Jacob A. Hassan¶, Carlos R. Morales¶,and Alexey V. Pshezhetsky‡�

From the ‡Department of Medical Genetics, Sainte-Justine Hospital, University of Montreal, Montreal, Quebec H3T 1C5and the ¶Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University, Montreal,Quebec H3A 2B2, Canada

Three different mammalian sialidases have been de-scribed as follows: lysosomal (Neu1, gene NEU1), cyto-plasmic (Neu2, gene NEU2), and plasma membrane(Neu3, gene NEU3). Because of mutations in the NEU1gene, the inherited deficiency of Neu1 in humans causesthe severe multisystemic neurodegenerative disordersialidosis. Galactosialidosis, a clinically similar disor-der, is caused by the secondary Neu1 deficiency becauseof genetic defects in cathepsin A that form a complexwith Neu1 and activate it. In this study we describe anovel lysosomal lumen sialidase encoded by the NEU4gene on human chromosome 2. We demonstrate thatNeu4 is ubiquitously expressed in human tissues andhas broad substrate specificity by being active againstsialylated oligosaccharides, glycoproteins, and ganglio-sides. In contrast to Neu1, Neu4 is targeted to lysosomesby the mannose 6-phospate receptor and does not re-quire association with other proteins for enzymatic ac-tivity. Expression of Neu4 in the cells of sialidosisand galactosialidosis patients results in clearance ofstorage materials from lysosomes suggesting that Neu4may be useful for developing new therapies for theseconditions.

Sialidases or neuraminidases are glycohydrolytic enzymesthat remove terminal sialic acid residues from sialylated gly-coproteins, oligosaccharides, and glycolipids. Three differentmammalian sialidases have been described as follows: lysoso-mal (Neu1, gene NEU1), cytoplasmic (Neu2, gene NEU2), andplasma membrane (Neu3, gene NEU3). Neu1 shows the high-est activity against oligosaccharides and short glycopeptides (1)and is involved in lysosomal catabolism of sialylated glycocon-jugates (reviewed in Ref. 2). Neu2 is active against �2–3-sialyl-ated oligosaccharides, glycopeptides, and gangliosides (3–5).The exact biological role of this enzyme is not known, but it was

suggested to cleave GM3 ganglioside, associated with the cy-toskeleton, leading to the alteration of cytoskeletal functions (6,7). In accordance with this, the Neu2 activity of melanoma cellsinversely correlates with their invasive and metastatic poten-tial (8). Neu3 is an integral membrane protein localized incaveolae microdomains of plasma membranes (9, 10). It isactive mostly against gangliosides involved in signal transduc-tion including GM1, GD1a, and other polysialogangliosides (11).Neu3 is probably involved in the modulation of the oligosac-charide chains of gangliosides on the cell surface in the courseof transformation, differentiation, and formation of cell con-tacts (12, 13). In particular it is implicated in cell signalingduring neuritogenesis (14), carcinogenesis, and apoptosis (15)as well as in insulin signaling (16).

The autosomal recessive disorder sialidosis (OMIM 256550)is caused by lysosomal sialidase deficiency because of muta-tions in the NEU1 gene (reviewed in Refs. 17 and 18). Anotherdisorder, galactosialidosis (OMIM 256540), is caused by thesecondary Neu1 deficiency because of genetic defects in cathep-sin A that form a complex with Neu1 and activate it (reviewedin Ref. 19). Both disorders belong to the group of lysosomalstorage diseases because sialidase deficiency in both cases dis-rupts the catabolic pathways for degradation of sialylated gly-coconjugates, causing their accumulation in the lysosome andexcretion in urine (17, 19). Both sialidosis and galactosialidosisinclude patients with severe early onset form and relativelymild late onset form. All patients develop visual defects, myoc-lonus syndrome, cherry-red macular spots, ataxia, hyper-re-flexia, and seizures. The severe early onset form is also asso-ciated with dysostosis multiplex, Hurler-like phenotype,mental and motor retardation, and hepatosplenomegaly (17,19). Identification of the human sialidase cDNA and gene onhuman chromosome 6 (20–22) paved the way for the charac-terization of the molecular basis of sialidosis, molecular diag-nostics of this disease, and genotype-phenotype correlations(reviewed in Ref. 18). However, development of enzyme re-placement or gene replacement therapies for sialidosis hasbeen hampered because Neu1 is an integral membrane enzyme(23) and requires co-expression with cathepsin A for inductionof enzymatic activity (20).

Here we describe that a novel human sialidase, Neu4 en-coded by a NEU4 gene on chromosome 2, exhibits broad sub-strate specificity and trafficking to the lysosomal lumen. Over-expression of Neu4 clears storage materials from culturedfibroblasts of sialidosis and galactosialidosis patients. Admin-istration of Neu4 and/or induction of NEU4 gene may thereforeoffer potential for the treatment of the severe multisystemicneurodegenerative disorders caused by defects of the NEU1gene.

* This work was supported in part by Operating Grants FRN 15079and MT-38107 from the Canadian Institutes of Health Research, Ge-nome Quebec Grant G202504, and by an equipment grant from theCanadian Foundation for Innovation (to A. V. P.). The costs of publica-tion of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s)NM_080741, NM_000434, NP_005374, and Q9UQ49.

§ Recipient of a postdoctoral fellowship from the Fonds de la Recher-che en Sante du Quebec and Fondation de l’Hopital Sainte-Justine.

� National Investigator of Fonds de la Recherche en Sante du Quebec.To whom correspondence should be addressed: Service de GenetiqueMedicale, Hopital Sainte-Justine, 3175 Cote Ste-Catherine, Montreal,Quebec H3T 1C5, Canada. Tel.: 514-345-4931 (ext. 2736); Fax: 514-345-4801; E-mail: alexei.pchejetski@umontreal.ca.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 35, Issue of August 27, pp. 37021–37029, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 37021

FIG. 1. Catalytic activity of Neu4. a, partial amino acid sequence alignment of human Neu4 (residues 16–480) with homologous sialidasesfrom Vibrio cholerae (KIT, GenBankTM accession number NP_231419), Micromonospora viridifaciens (EUR, accession number A45244), Salmo-nella typhimurium (SIL, accession number AAL19864), as well as human cytosolic (Neu2, accession number, NP_005374, residues 6–379) andlysosomal sialidases (Neu1, accession number NM_000434, residues 61–415). The identical residues are boxed. Active site residues are shown asblack on yellow and Asp box repeats as black on blue. The �-sheets in the structures of bacterial sialidases are indicated by arrows above thealignment. b, pH dependence of the maximal reaction rate of the 4-MU-NANA hydrolysis catalyzed by Neu1, Neu3, and Neu4. Human Neu1, Neu3,and Neu4 were expressed in COS-7 cells, and their activity was measured as described under “Materials and Methods.” Data represent the mean �S.E. of three independent experiments. c, substrate specificity of human Neu1, Neu3, and Neu4. Enzymatic activity of human Neu1, Neu3, andNeu4 expressed in COS-7 cells against bovine mucin, sialyllactose, 4-MU-NANA, and mixed bovine gangliosides was measured at pH optimum foreach enzyme as described under “Materials and Methods.”

Lysosomal Lumen Sialidase Neu437022

MATERIALS AND METHODS

Plasmids—The human Neu4 cDNA clone 4156395 was obtained fromthe ATCC (American Type Culture Collection, Manassas, VA). For theexpression of Neu4-GFP and GFP-Neu4 fusion proteins, an entirecDNA was subcloned into EcoRI-BamHI sites of pEGFP_N3 and pEG-FP_C1 vectors (Clontech). For the expression of Neu4-FLAG fusionprotein, the Neu4 cDNA was subcloned into EcoRI-XhoI sites of pCMV-Tag4A vector (Stratagene, La Jolla, CA). Neu3 cDNA, kindly providedby Dr. Miyagi from the Research Institute, Miyagi Prefectural CancerCenter (Japan), was subcloned into SacII-XhoI sites of pCMV-Scriptvector (Stratagene). Expression constructs for human lysosomal siali-dase Neu1 (pCMV-Neu1) and human lysosomal cathepsin A (pCMV-CathA) were described before (22).

Expression of Neu4 in Human Fibroblasts and in COS-7 Cells—Human skin fibroblasts of sialidosis, galactosialidosis, and mucolipido-sis II patients and of normal controls were obtained from NIGMSHuman Genetic Mutant Cell Repository (GM2438A), Montreal Chil-dren’s Hospital Cell Repository (WG0544), and from Ste-Justine Hos-pital cell repository (7615).

Skin fibroblasts cultured in Eagle’s minimal essential medium (In-vitrogen), supplemented with 10% (v/v) fetal calf serum (Invitrogen)and antibiotics, were trypsinized, suspended at a density of 6 � 106 cellsper ml in Eagle’s minimal essential medium, and electroporated byusing a Gene Pulser II (Bio-Rad). COS-7 cells cultured until 70% con-fluency in Eagle’s minimal essential medium supplemented with 10%(v/v) fetal calf serum (Invitrogen) and antibiotics were transfected byusing a LipofectAMINE Plus kit (Invitrogen) according to the protocol ofthe manufacturer.

Subcellular Fractionation of COS-7 Cells—Subcellular fractionationwas performed as described for rat liver (24), but a Dounce tissuegrinder was used to break the cells instead of a Potter-Elvehjem ho-mogenizer. Light mitochondrial fraction was further separated usingthe density gradient ultracentrifugation. The fraction was applied atthe bottom of a 0–22% OptiPrep (Invitrogen) gradient. After a 2-hcentrifugation at 24,000 rpm in an SW41-Ti Beckman rotor, 12 frac-tions were collected from the top to the bottom of the tube.

Enzyme Assays—Sialidase, �-galactosidase, and �-hexosaminidaseactivities in cellular homogenates and in subcellular fractions wereassayed by using the corresponding fluorogenic 4-methylumbelliferylglycoside substrates (25, 26). Alkaline phosphatase, glutamate dehy-drogenase, and esterase in subcellular factions were measured as de-scribed elsewhere (27–29). Sialidase activity toward mucin, sialyllac-tose, and mixed bovine gangliosides was measured as described (1, 11)in the presence of Triton X-100 (0.2%) or sodium deoxycholate (0.1%).The concentration of released sialic acid was measured by the thiobar-bituric method (31). Enzyme activity is expressed as the conversion of 1nmol of substrate per h. Protein concentration was assayed according toBradford (32).

Confocal Immunofluorescence Microscopy—Skin fibroblasts orCOS-7 cells were treated for 45 min with 75 nM LysoTracker RedDND-99 dye (Molecular Probes, Eugene, OR), washed twice with ice-cold PBS,1 and fixed with 3.8% paraformaldehyde in PBS for 30 min.Cells were permeabilized by 0.5% Triton X-100, washed twice with PBS,stained with monoclonal anti-FLAG antibodies, and counterstainedwith Oregon Green 488-conjugated anti-mouse IgG antibodies (Molec-ular Probes, Eugene, OR). Alternatively, the cells were double stained

with anti-FLAG antibodies and Texas Red-labeled monoclonal antibod-ies against LAMP-2 (Washington Biotechnology Inc., Baltimore, MD).Slides were studied on a Zeiss LSM510 inverted confocal microscope(Zeiss).

Electron Microscopy—The fibroblasts cell lines were trypsinized, pel-leted, and fixed for 1 h with 2.5% glutaraldehyde in 0.1 M phosphatebuffer. After embedding in 2% agarose, the cells were post-fixed withosmium ferrocyanide. Increasing concentrations of ethanol were usedfor subsequent dehydration. The cells were then embedded in Epon.Semithin sections were cut and mounted on 200 mesh copper grids.Staining of the grids was done with uranyl acetate for 5 min followed bylead citrate for 2 min as described (33–35). The grids were viewed on aJEOL JEM-2000 FX electron microscope. Lysosomes were identified byusing the following morphological criteria: lysosomes have to be spher-ical, ranging from 0.2 to 0.4 �m in diameter, and have moderate tostrong electron density (33, 34). Sometimes secretory granules may fallwithin this morphological description. However, the employed cell lineused in this investigation was derived from fibroblasts, which do notcontain secretory granules.

For both Neu1 and Neu4-transfected fibroblasts, �100 cells per grid(n � 3) were counted and classified according to the characteristic oftheir lysosomes as rescued or nonrescued cells. In the case of thegalactosialidosis cell line transfected with the Neu4 construct, it wasalso possible to identify and count partially rescued cells.

Metabolic Labeling of Neu4 with [32P]Phosphate—Twenty-four hoursafter transfection with Neu4-FLAG plasmid, COS-7 cells were incu-bated for 15 min in phosphate-free Dulbecco’s modified Eagle’s medium(Invitrogen) and for 3 h in the same medium supplemented with[32P]phosphate (ICN, Irvine, CA), 0.1 mCi/ml. The radioactive mediumwas then removed. Cells were placed on ice, washed twice with ice-coldPBS, and lysed for 30 min in a buffer containing 50 mM Tris-HCl, pH7.4, 150 mM NaCl,1 mM EDTA, 1% (v/v) Triton X-100, 1 mM �-toluene-sulfonyl fluoride, 50 mM sodium orthovanadate, 50 mM NaF, 1.5 mM

MgCl2, and complete protease inhibitor mixture (Sigma). The lysatewas collected and centrifuged at 12,000 � g for 10 min to remove the celldebris. The purification of Neu4-FLAG on the immunoaffinity gel(Sigma) was done as recommended by the manufacturer. Neu4-FLAGwas eluted with the 100 �g/ml solution of the FLAG peptide (Sigma) inTris-buffered saline. The eluted fraction was analyzed by SDS-PAGEfollowed by silver staining and autoradiographic detection. Detectablebands were excised for MS analysis. A second sample prior to SDSanalysis was deglycosylated with endoglycosidase H. EndoglycosidaseH (Sigma) was added to the sample in a final concentration of 50milliunits/ml, and the mixture was incubated overnight at 37 °C.

MS Identification—In-gel digestion of proteins and extraction of pep-tides was performed as described (36). Samples were analyzed by anLC-MS/MS system consisting of a nanoflow liquid chromatographysystem and an ion trap 1100 series LC MSD mass spectrometer (Nano-Flow Proteomics Solution, Agilent Technologies, Santa Clara, CA). Pep-tides were separated by reversed phase high pressure liquid chroma-tography on a Zorbax 300SB-C18 column (Agilent) with a gradient of3–90% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min. Thecolumn eluent was sprayed directly into the mass spectrometer. Spectrawere searched against NCBI NR data base (NCBI, Bethesda, MD) usinga Spectrum Mill software (Agilent).

Northern Blot Analysis—A 12-lane MTN blot containing 1 �g ofpoly(A)� RNA from various human tissues per lane (Clontech) washybridized with the following probes: a 500-bp Neu4 cDNA fragmentobtained from clone 4156395 by PvuII digestion, an entire Neu1 cDNA,a 700-bp Neu3 fragment obtained from a cDNA clone by PstI digestion,and an entire cDNA of human �-actin. All probes were labeled with

1 The abbreviations used are: PBS, phosphate-buffered saline; MS,mass spectrometry; MS/MS, tandem mass spectrometry; LC, liquidchromatography; 4-MU-NANA, 2�-(4-methylumbelliferyl)-�-D-N-acetyl-neuraminic acid; GFP, green fluorescent protein.

FIG. 1—continued

Lysosomal Lumen Sialidase Neu4 37023

[32P]dCTP by random priming using a MegaPrime labeling kit (Amer-sham Biosciences). Pre-hybridization of the blots was performed at68 °C for 30 min in ExpressHybTM (Clontech). The denatured probeswere added directly to the pre-hybridization solution and incubated at68 °C for 1 h. The blots were washed twice for 30 min with 2� SSC,0.05% SDS at room temperature, once for 40 min with 0.1� SSC, 0.1%SDS at 50 °C, and exposed to a BioMax film overnight at �80 °C.

RESULTS

Sialidase Activity of Neu4—Sequencing and annotation ofthe human genome predicted the presence on chromosome 2 ofa gene presumably coding for another sialidase (NEU4) with anamino acid sequence homology to Neu1, Neu2, and Neu3. Ourrecent experiments aimed on analysis of the proteome of ratliver lysosome have identified MS and MS/MS spectra of thepeptide VPALLCVPPRPTLLAFAEQR derived from the se-quence of gene product XP_237421 (GI:34877834), the rat an-alog of human NEU4.2 These results suggested that NEU4 isexpressed at least in rat liver and inspired further studies ofthis protein. The alignment of the human Neu4 amino acidsequence with those of crystallized bacterial sialidases (Fig. 1a)demonstrated conservation in Neu4 of all important active siteresidues, which bind the carboxylate group of the sialic acidsubstrate (Arg-35, -254, and -401) as well as N-acetyl-/N-glyco-lyl-binding active site residues (Asn-98) and suggested thatNeu4 may have sialidase activity. The topology of secondarystructural elements such as antiparallel �-strands or the so-called “Asp boxes” (Ser/Thr-X-Asp-X-Gly-X-X-Trp/Phe) is alsoconserved in Neu4 (Fig. 1a). Asp boxes are found in all bacterialand mammalian sialidases always on the turns between �Dand �E, �H and �I, �N and �O, and �S and �T, i.e. between thethird and the fourth �-strand of each sheet. All Asp boxes havesimilar conformation with the aromatic residues packed intothe hydrophobic core that stabilizes the turn, whereas thehydrophilic Asp residues are always exposed to the solvent.Neu4 therefore most probably shares general “sialidase fold”consisting of six four-stranded antiparallel �-sheets arrangedas the blades of a propeller around a pseudo 6-fold axis (37).

By using the synthetic fluorescent substrate 2�-(4-methylum-belliferyl)-�-D-N-acetylneuraminic acid (4-MU-NANA), we dem-onstrated that the NEU4 gene product indeed had sialidaseactivity. COS-7 cells transfected with the Neu4 cDNA showed a10-fold increase in sialidase activity compared with mock-trans-fected cells. The pH-optimum was 3.5, i.e. close to that of Neu1and Neu3 but in contrast to those enzymes Neu4 retained almost40% of its maximal activity at higher pH (Fig. 1b). Although allmammalian sialidases show activity against 4-MU-NANA, theydiffer in their specificity against natural substrates, Neu1 beingmost active on oligosaccharides and Neu2 and Neu3 on glycolip-ids (16–20). Human Neu4 showed broad substrate specificity(Fig. 1c), being almost equally active on glycoproteins (mucin),

2 K. Landry, M. Ashmarina, and A. V. Pshezhetsky, unpublishedobservations.

FIG. 2. Intralysosomal location of Neu4. Co-localization of Neu4-GFP (a) and Neu4-FLAG (b) fusion proteins with the lysosomal marker,LysoTracker Red. COS-7 cells (a) and cultured human skin fibroblasts(b) were transfected with Neu4-GFP and Neu4-FLAG as described.

Slides were studied on a Zeiss LSM510 inverted confocal microscope.Magnification �600. c, distribution of marker enzymes, lysosomal�-hexosaminidase (HEX) and mitochondrial glutamate dehydrogenase(GDH), and Neu4 after the differential centrifugation of COS-7 cellhomogenate. Data represent the mean � S.E. of three independentexperiments. Values shown are percentage of enzyme activity recoveredin each fraction as compared with a total homogenate. Nuc, nuclearfraction; HM, heavy mitochondrial fraction; LM, light mitochondrialfraction; MS, microsomal fraction; Cyt, cytosol. d, equilibrium centrif-ugation in a density gradient of light mitochondrial fraction from COS-7cells. The x axis represents the density gradient fractions, with fraction1 being the least dense. The ordinate represents relative activity of theenzymes indicated in each panel. The locations of other marker en-zymes (not shown) are as follows: esterase, fractions 3–5; �-galactosid-ase, fractions 7–9.

Lysosomal Lumen Sialidase Neu437024

FIG. 3. Phosphorylation of Neu4. COS-7 cells transfected with Neu4-FLAG were labeled with [32P]phosphate as described under “Materialsand Methods.” Proteins purified from the cell lysate on anti-FLAG immunoaffinity column treated (�EH) or not treated with endoglycosidase H(�EH) were resolved by SDS-PAGE. Gels were analyzed by autoradiography (a and d), electrotransfered to a nitrocellulose membrane, and stainedwith mouse monoclonal anti-FLAG antibodies followed by chemiluminescence detection (b) or stained with silver (c). A 60-kDa phosphorylatedprotein band was identified as Neu4 (e). Upper panel, tandem mass spectra of the doubly protonated precursor ion with m/z of 911.1 obtained fromnano-LC-MS analysis of a 60-kDa band. Lower panel, peptides identified by Spectrum Mill software, their scores, and location in the Neu4sequence.

Lysosomal Lumen Sialidase Neu4 37025

oligosaccharides (sialyllactose), 4-MU-NANA, and sialylated gly-colipids (mixed bovine gangliosides).

Intracellular Localization and Trafficking of Neu4—Our re-sults indicate that Neu4 localizes to lysosomes. First, confocalfluorescent microscopy showed that Neu4 tagged with GFP orFLAG peptides was targeted in COS-7 cells and human fibro-blasts to cytoplasmic organelles co-localizing with the lysoso-mal markers LysoTracker Red (Fig. 2, a and b) or LAMP-2 (notshown). Second, the majority of Neu4-related sialidase activityin the COS-7 cells transfected with pCMV-SPORT-Neu4 wasfound in the light mitochondrial fraction-enriched in lysosomesand containing most of the lysosomal �-hexosaminidase activ-ity (Fig. 2c). When the light mitochondrial fraction was furtheranalyzed by density gradient ultracentrifugation, Neu4 activ-ity was found exclusively in the fractions containing lysosomesand marked by the presence of �-hexosaminidase (Fig. 2d) and�-galactosidase (not shown).

97% of the Neu4-related sialidase activity in COS-7 cellstransfected with Neu4-FLAG cDNA was found in the superna-tant after the centrifugation of the sonicated cell homogenateat 100,000 � g for 1 h (not shown). These data suggest that incontrast to an integral lysosomal membrane protein, Neu1,which cannot be solubilized without detergent (23), Neu4 is asoluble hydrolase located in the lysosomal lumen. This is alsoconsistent with the results of computer analysis of the Neu4amino acid sequence by the Classification and SecondaryStructure Prediction of Membrane Proteins algorithm (SOSUIsystem, Department of Biotechnology, Tokyo Universityof Agriculture and Technology; sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html) which predicted that Neu4 is a soluble pro-tein, whereas Neu1 and Neu3 both contain transmembranehelixes. With rare exceptions, precursors of lysosomal luminalproteins are targeted by the mannose 6-phosphate receptor.Our data demonstrate that Neu4 is also transported by thismechanism. Neu4 binds to a concanavalin A-Sepharose (notshown) suggesting that it is glycosylated. To detect if Neu4precursor is phosphorylated, we performed 32P-labeling ofCOS-7 cells transfected with FLAG-tagged Neu4. Proteins pu-rified from the cell lysate on anti-FLAG immunoaffinity col-umn were analyzed by SDS-PAGE with autoradiographic de-tection. Altogether we found five phosphorylated protein bands(Fig. 3a, Neu4). Three of those bands were also present in theimmunopurified fraction of mock-transfected cells (Fig. 3a,Mock) and probably represented phosphorylated proteins non-specifically absorbed on the matrix. Of the other two bands, theupper band (60 kDa) cross-reacted with anti-FLAG antibodies(Fig. 3b). We therefore excised it from gel (Fig. 3c), digestedwith trypsin, and analyzed tryptic peptides by tandem massspectrometry (MS/MS). MS/MS identified the 60-kDa band ashuman Neu4 (gi21704287) with 18% sequence coverage (Fig.3d). When the immunopurified sample was treated with en-doglycosidase H, the phosphorylation of Neu4 was not detectedshowing that the phosphate label is on the oligosaccharideportion of the enzyme rather then on polypeptide chain(Fig. 3c).

Further evidence that Neu4 is targeted by the mannose6-phosphate receptor was obtained when we found that it isdeficient in mucolipidosis II cells (ML II, OMIM 252500). ML IIis caused by genetic deficiency of UDP-GlcNAc:lysosomal en-zyme N-acetylglucosamine-1-phosphotransferase, which is nec-essary for generation of mannose 6-phosphate signal in precur-sors of soluble lysosomal enzymes (38). Transfection withNeu4-FLAG cDNA did not increase sialidase activity in the MLII fibroblasts in contrast to the cells from sialidosis and galac-tosialidosis patients (Table I). Moreover, by Western blotFLAG-Neu4 peptide was absent from the cell homogenate butdetected in the culture medium together with the precursors ofother soluble lysosomal enzymes, secreted out of the ML II cellsinstead of being targeted to the lysosome (not shown). Similarresults were also obtained for GFP-Neu4 fusion protein. Be-cause we could not detect sialidase activity in the culturemedium of ML II fibroblasts, we assumed that the Neu4 pre-cursor probably had to be activated in the lysosome.

Expression of Neu4 in Human Tissues—Northern blotshowed that similarly to NEU1, NEU4 is a ubiquitously ex-pressed housekeeping gene found in all tissues studied (Fig. 4).However, the expression profiles of NEU1 and NEU4 do notcompletely coincide. High expression of both NEU1 and NEU4is detected in skeletal muscle, heart, placenta, and liver,whereas NEU1 is expressed at a much higher level in kidney,spleen, thymus, colon, brain, lung, and small intestine (Fig. 4).The specific expression patterns of the two lysosomal sialidasesmerit further attention because they may reflect other as yetuncharacterized functions not necessarily related to lysosomalcatabolism.

Neu4 Confers Normal Phenotype to Sialidosis Cells—Theobjective of this study was to determine whether Neu4 cansubstitute Neu1 in the lysosomal catabolism of sialylated gly-coconjugates. We have examined the lysosomal storage in thecells of a sialidosis patient (line WG0544) transfected withNeu4-expressing plasmids. As a positive control the same cellline was transfected with Neu1, and as a negative control thecells were transfected with the plasmid alone (mock cells). Inaddition, a second negative control composed of nontransfectedcells from the same sialidosis cell line was used.

When we examined the morphological phenotypes of thelysosomal compartments of sialidosis cells and of those trans-fected with the Neu4 expression vector by electron microscopy,we found that transfection with the Neu4 expression vectorresulted in clear improvement of lysosomal storage. Nontrans-fected and mock-transfected cells presented an accumulation oflarge, pale, spherical vacuoles (0.2–1.5 �m in diameter) typicalof lysosomes of cells with storage disorders (Fig. 5, a and b).These abnormal lysosomes sometimes presented membranous

FIG. 4. Northern blot analysis of Neu4 mRNA in various hu-man tissues. A 12-lane blot containing 1 �g of poly(A)� RNA fromvarious adult human tissues per lane was hybridized with 32P-labeledprobes for human Neu1, Neu3, Neu4, or �-actin as described under“Materials and Methods.” By using a total 32P-labeled Neu4 cDNA as aprobe, a 3.6-kb transcript was also detected in the liver.

TABLE ISialidase activity in human skin fibroblasts from sialidosis,

galactosialidosis, and ML II patients transfected withpCMV-Neu4-FLAG and pCMV-Tag4a plasmids

Data represent the mean � S.E. of three independent experiments.

PlasmidEnzyme activity

Sialidosis Galactosialidosis ML II

nmol/h mg protein

pCMV-Tag4a 0.4 � 0.5 1.1 � 0.9 1.4 � 0.7

pCMV-Neu4-FLAG 12 � 5.5 16 � 7.3 0.4 � 1.2

Lysosomal Lumen Sialidase Neu437026

profiles and a peripheral rim of electron-dense material. Incontrast, cells transfected with the Neu4 and Neu1 presentedsmall electron-dense granules (0.2–0.4 �m in diameter) typicalof normal lysosomes (Fig. 5, c and d). Both Neu4 and Neu1therefore conferred normal lysosomal morphology to sialidosisfibroblasts, suggesting that Neu4, similarly to Neu1, is activeon undigested substrates containing neuraminic acid. Theseobservations correlated well with the targeting of Neu4 to thelysosomes of transfected cells as shown by our immunofluores-cence studies (Fig. 2, a and b). Up to 55% (� 4 S.D.) of the cellsshowed normal phenotype, whereas only 3–5% of them weretransfected and expressed Neu4 protein according to immuno-histochemical staining with anti-FLAG antibodies (not shown),

suggesting that Neu4 secreted by the transfected cells is endo-cytosed into adjacent nontransfected cells via mannose 6-phos-phate receptors on their surface. A very small amount of inter-mediate cells was observed in these experiments.

Fibroblasts of galactosialidosis patient (line GM2438A)transfected with the Neu4 construct exhibited three distinctphenotypes. Approximately 39% (� 3 S.D.) of the cells pre-sented accumulation of large, pale, spherical vacuoles (0.2 to1.5 �m in diameter) sometimes containing intramembranousprofiles typical of lysosomes with storage disorders (Fig. 6, aand d). However, 25% (� 4 S.D.) of the Neu4-transfected cellspresented small electron-dense granules (0.2–04 �m in diam-eter) typical of normal lysosomes (Fig. 6, c and f). The remain-

FIG. 5. Electron micrographs of representative sialidosis fibroblasts from each of the four experimental groups: nontransfectedcell (a); pCMV-Tag4a (mock)-transfected cell (b); pCMV-Neu4-FLAG-transfected cell (c); and pCMV-Neu1-transfected cell (d).Although cells in a and b (negative controls) present an accumulation of large pale vacuoles, cells in c and d exhibit small electron-dense granularbodies. Magnification �10,000.

Lysosomal Lumen Sialidase Neu4 37027

ing 36% (� 5 S.D.) represented cells with intermediate lyso-somes containing electron-lucent and electron-dense materialand membranous profiles (Fig. 6, b and e).

DISCUSSION

Our data demonstrate that the product of the NEU4 gene onhuman chromosome 2 is a novel lysosomal lumen sialidase thatshows activity against 4-MU-NANA and acidic pH optimum.While this paper was in preparation, the NEU4 gene productwas reported by two other groups (39, 40), who also docu-mented catalytic activity against the synthetic substrate. How-ever, in contrast to these studies, we show that recombinantNeu4 expressed in COS-7 cells is active against all major typesof sialylated conjugates including oligosaccharides, glycopro-teins, and glycolipids. High activity of Neu4 against ganglio-sides is of particular interest because it may explain an appar-ent longstanding paradox. Several laboratories reported (11,41–43) that Neu1 is able to catalyze the hydrolysis of ganglio-sides in the presence of bile salts or the sphingolipid activatorprotein saposin B. However, the analysis of storage products insialidosis and galactosialidosis patients (17) or in the knockoutmouse model of galactosialidosis (44) did not show storage ofgangliosides, suggesting that Neu1 is not essential for theircatabolism. Although Neu2 and Neu3 desialylate glycolipids invitro (5, 45), they cannot account for ganglioside catabolismbecause they are not present in the lysosome. Our data showthat Neu1 expressed in COS-7 cells (Fig. 1c) or purified fromhuman placenta (not shown) has very little activity towardmixed gangliosides even in the presence of bile salts. In con-trast, mixed bovine gangliosides are desialylated by Neu4 at arate compatible to that of 4-MU-NANA (Fig. 1c), suggestingthat Neu4 is the enzyme responsible for the catabolism ofsialylated glycolipids. In vitro, the reaction requires a deter-

gent, but in the cell, gangliosides could probably be hydrolyzedin the presence of sphingolipid activator proteins. Other exper-iments, in particular those involving Neu4 knock-out models,are required to define the biological role of Neu4 and to provethat it acts on gangliosides in lysosomes. Such experiments arecurrently in progress in our laboratory.

Our data also showed that Neu4 is active against a majorityof endogenous substrates of Neu1. Being expressed in Neu1-deficient sialidosis fibroblasts, Neu4 completely eliminated un-digested substrates of Neu1 and restored normal morphologicalphenotype of the lysosomal compartment, thus offering thera-peutic potential. The strategy for treatment of lysosomal stor-age disorders (enzyme replacement therapy, bone marrowtransplantation, or gene therapy) relies on the principle of“cross-correction” where precursors of missing enzymes se-creted from donor cells or exogenously supplied are internal-ized by other cells through the receptor-mediated endocytosis(30). Therefore, the therapeutic success for a particular disor-der depends on the molecular properties of the deficient en-zyme as follows: its solubility and stability, mechanism of itslysosomal targeting, whether it has to be post-translationallymodified, etc. In this respect disorders caused by inheritedsialidase deficiency did not have much perspective becauseNeu1 is an integral membrane protein that cannot be secretedfrom donor cells (23) and requires co-expression with cathepsinA for activation and stabilization (20, 22). In contrast, Neu4 isa soluble enzyme, whose precursor is targeted to the lysosomeby the mannose 6-phospate receptor and can be potentiallytaken up by the cells from the medium. In accordance with thiswe observed that complete elimination of storage materialshappened in 55% of sialidosis cells and in 25% of galactosia-lidosis cells (in addition 36% of galactosialidosis cells showed

FIG. 6. Electron micrographs of representative galactosialidosis fibroblasts transfected with pCMV-Neu4-FLAG vector. a, non-transfected fibroblast presenting accumulation of large pale vacuoles (�15,000). At higher magnification (d) these vacuoles sometimes containedundigested membranes (�25,000). b, cell presenting partially rescued lysosomes (�15,000). At higher magnification (e) these lysosomes containintramembranous profiles and electron-dense material. c, rescued cell exhibiting normal-looking lysosomes. (�15,000). At higher magnification (f)the lysosomes contain a homogenous electron-dense material (�25,000).

Lysosomal Lumen Sialidase Neu437028

partially corrected phenotype), whereas only 3–5% of cells weretransfected with Neu4 plasmid as assayed by immunohisto-chemistry. These data show that the Neu4 released from thetransfected cells enters cells neighboring the Neu4-expressingcells and corrects their phenotype. Therefore, recombinant hu-man Neu4 might be of potential use for enzyme replacementtherapy in sialidosis and galactosialidosis. However, enzymereplacement rarely achieves superphysiologic levels of the en-zyme in target tissues, and because patients with Neu1 defi-ciency but with two normal Neu4 alleles still develop the dis-ease, physiologic levels of Neu4 are likely not sufficient toprevent accumulation of sialylated compounds. Much more at-tractive, therefore, would be to induce the expression of theendogenous NEU4 gene to compensate for NEU1 deficiency.Northern blotting revealed Neu4 expression in every humantissue examined (Fig. 4) suggesting that such an approach mayhave a general effect throughout the whole organism, includingthe central nervous system, which is presently beyond thescope of the enzyme replacement therapy.

Acknowledgments—We thank Dr. T. Miyagi for providing a humanNeu3 cDNA clone. We also thank Liliane Gallant for help in prepara-tion of the manuscript and Grant Mitchell, Jacques Michaud, andMila Ashmarina for helpful advice.

REFERENCES

1. Miyagi, T., Hata, K., Hasegawa, A., and Aoyagi, T. (1993) Glycoconj. J. 10,45–49

2. Pshezhetsky, A. V., and Ashmarina, M. (2001) Prog. Nucleic Acids Res. Mol.Biol. 69, 81–114

3. Miyagi, T., and Tsuiki, S. (1985) J. Biol. Chem. 260, 710–67164. Monti, E., Preti, A., Nesti, C., Ballabio, A., and Borsani, G. (1999) Glycobiology

9, 1313–13215. Tringali, C., Papini, N., Fusi, P., Croci, G., Borsani, G., Preti, A., Tortora, P.,

Tettamanti, G., Venerando, B., and Monti, E. (2004) J. Biol. Chem. 279,3169–3179

6. Akita, H., Miyagi, T., Hata, K., and Kagayama, M. (1997) Histochem. Cell Biol.107, 495–503

7. Sato, K., and Miyagi, T. (1996) Biochem. Biophys. Res. Commun. 221, 826–8308. Tokuyama, S., Moriya, S., Taniguchi, S., Yasui, A., Miyazaki, J., Orikasa, S.,

and Miyagi, T. (1997) Int. J. Cancer 73, 410–4159. Wada, T., Yoshikawa, Y., Tokuyama, S., Kuwabara, M., Akita, H., and Miyagi,

T. (1999) Biochem. Biophys. Res. Commun. 261, 21–2710. Wang, Y., Yamaguchi, K., Wada, T., Hata, K., Zhao, X., Fujimoto, T., and

Miyagi, T. (2002) J. Biol. Chem. 277, 26252–2625911. Schneider-Jakob, H. R., and Cantz, M. (1991) Biol. Chem. Hoppe-Seyler 372,

443–45012. Kopitz, J., von Reitzenstein, C., Sinz, K., and Cantz, M. (1996) Glycobiology 6,

367–37613. Kopitz, J., von Reitzenstein, C., Burchert, M., Cantz, M., and Gabius, H. J.

(1998) J. Biol. Chem. 273, 11205–1121114. Wu, G., and Ledeen, R. W. (1991) J. Neurochem. 56, 95–10415. Kakugawa, Y., Wada, T., Yamaguchi, K., Yamanami, H., Ouchi, K., Sato, I.,

and Miyagi, T. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10718–1072316. Sasaki, A., Hata, K., Suzuki, S., Sawada, M., Wada, T., Yamaguchi, K., Obi-

nata, M., Tateno, H., Suzuki, H., and Miyagi, T. (2003) J. Biol. Chem. 278,27896–27902

17. Thomas, G. H. (2001) in Metabolic and Molecular Bases of Inherited Disease(Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) pp. 3507–3534,McGraw-Hill Inc., New York

18. Seyrantepe, V., Poupetova, H., Froissart, R., Zabot, M.-T., and Pshezhetsky,A. V. (2003) Hum. Mutat. 22, 343–352

19. d’Azzo, A., Andria, G., Strisciuglio, P., and Galijaard, H. (2001) in Metabolicand Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly,W. S., and Valle, D., eds) pp. 3811–3826, McGraw-Hill Inc., New York

20. Bonten, E., van der Spoel, S. A., Fornerod, M., Grosveld, G., and d’Azzo, A.(1996) Genes Dev. 10, 3156–3169

21. Milner, C. M., Smith, S. V., Carrillo, M. B., Taylor, G. L., Hollinshead, M., andCampbell, R. D. (1997) J. Biol. Chem. 272, 4549–4558

22. Pshezhetsky, A. V., Richard, C., Michaud, L., Igdoura, S., Wang, S., Elsliger,M. A., Qu, J., Leclerc, D., Gravel, R., Dallaire, L., and Potier, M. (1997) Nat.Genet. 15, 316–320

23. Lukong, K. E., Seyrantepe, V., Landry, K., Trudel, S., Ahmad, A., Gahl, W. A.,Lefrancois, S., Morales, C. R., and Pshezhetsky, A. V. (2001) J. Biol. Chem.276, 46172–46181

24. Graham, Y. (1984) in Centrifugation: A Practical Approach (Rickwood, D., ed)pp. 161–182, 2nd Ed., IRL Press at Oxford University Press, Oxford

25. Potier, M., Mameli, L., Belisle, M., Dallaire, L., and Melancon, S. B. (1979)Anal. Biochem. 94, 287–296

26. Rome, L. H., Garvin, A. J., Allietta, M. M., and Neufeld, E. F. (1979) Cell 17,143–153

27. Bergmeyer, H., Gawehn, K., and Grassl, M. (1974) in Methods of EnzymaticAnalysis (Bergmeyer, H. V., ed) pp. 495–496, Verlag Chemie, Weinheim,Germany

28. Schmidt, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed)pp. 650–656, Verlag Chemie, Weinheim, Germany

29. Burdette, R. A., and Quinn, D. M. (1986) J. Biol. Chem. 261, 12016–1202130. Neufeld, E. F. (1991) Annu. Rev. Biochem. 60, 257–28031. Manzi, A. E. (2003) in Current Protocols in Molecular Biology (Ausubel, F. M.,

Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., andStruhl, K., eds) Vol. 3, pp. 17.18.8–17.18.10, John Wiley & Sons, Inc., NewYork

32. Bradford, M. M. (1976) Anal. Biochem. 72, 248–25433. Igdoura, S. A., Rasky, A., and Morales, C. R. (1996) Cell Tissue Res. 283,

385–39434. Lefrancois, S., Michaud, L., Potier, M., Igdoura, S. A., and Morales, C. R.

(1999) J. Lipid Res. 40, 1593–160335. Lefrancois, S., May, T., Knight, C., Bourbeau, D., and Morales, C. R. (2002)

J. Biol. Chem. 277, 17188–1719936. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Anal. Chem. 68,

850–85837. Varghese, J. N., Laver, W. G., and Colman, P. M. (1983) Nature 303, 35–4038. Kornfeld, S., and Sly, W. S. (2001) in Metabolic and Molecular Bases of

Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D.,eds) pp. 3469–3482, McGraw-Hill Inc., New York

39. Comelli, E. M., Amado, M., Lustig, S. R., and Paulson, J. C. (2003) Gene (Amst.)321, 155–161

40. Monti, E., Bassi, M. T., Bresciani, R., Civini, S., Croci, G. L., Papini, N., Riboni,M., Zanchetti, G., Ballabio, A., Preti, A., Tettamanti, G., Venerando, B., andBorsani, G. (2004) Genomics 83, 445–453

41. Hiraiwa, M., Uda, Y., Nishizawa, M., and Miyatake, T. (1987) J. Biochem.(Tokyo) 101, 1273–1279

42. Hiraiwa, M., Nishizawa, M., Uda, Y., Nakajima, T., and Miyatake, T. (1988)J. Biochem. (Tokyo) 103, 86–90

43. Fingerhut, R., van der Horst, G. T., Verheijen, F. W., and Conzelmann, E.(1992) Eur. J. Biochem. 208, 623–629

44. Zhou, X. Y., Morreau, H., Rottier, R., Davis, D., Bonten, E., Gillemans, N.,Wenger, D., Grosveld, F. G., Doherty, P., Suzuki, K., Grosveld, G., andd’Azzo, A. (1995) Genes Dev. 9, 2623–2634

45. Li, S. C., Li, Y. T., Moriya, S., and Miyagi, T. (2001) Biochem. J. 360, 233–237

Lysosomal Lumen Sialidase Neu4 37029